Abstract The vast majority of HIV-1 infections occur at mucosal surfaces in the body. There is therefore an immediate need for potent HIV vaccines that can provide barrier protection at mucosal surfaces. While there is this need, most HIV vaccines have been developed and tested for their ability to drive systemic immune responses and not for mucosal responses. Given that systemic immunization generally does not provoke potent mucosal protection, this project will the ability of adenoviral vaccines to mediate protection against mucosal SIV infectoin after systemic immunization and compare this to protection after mucosal immunization by the oral route. Current first generation adenoviral (FG-Ad) vaccines encode 17 adenoviral open reading frames that can be targeted by anti-vector T cells. In contrast, helper-dependent adenoviral (HD-Ad) vectors encode zero. HD-Ad vectors therefore have a safety advantage over current clinically-utilized FG-Ad vaccines. Helper-dependent vectors can also be serotype-switched to evade pre-existing and vector-induced immune responses allowing four or more rounds of immunization. Given this and the recent side effects observed in the HIV STEP vaccine trial, this project will develop HD-Ad vectors to evade pre-existing immunity in humans. This project will first compare the ability of serotype-switched HD-Ad vectors to drive anti-SIV immune responses and protect macaques from mucosal SIV challenge after intramuscular and oral immunization. The ability of the serotype-switched vectors to evade anti-vector immune responses will be compared to the stealth abilities of helper-dependent adenovirus that is shielded with PEG. These vaccine challenge studies will be complemented with aims geared to improve the functionality of both the HD-Ad vectors and PEG with the goal of better evading immune responses in humans. These improvements will be made by genetic and chemical engineering and will be tested for function in mouse models. Development of these technologies combined with comparisons made in the macaque-SIV mucosal challenge model will provide information and reagents relevant to translation of these vaccines into humans. This work will also generate adenoviral vaccines with improved safety and efficacy as compared to current adenoviral vaccines in the clinic.